Fluorescent magnetic nanoparticles and process of preparation

ABSTRACT

This invention provides nanometer-sized fluorescent magnetic particles and processes of making them. The nanoparticle has a core particle comprising a magnetic material and a fluorescent material, and the particle size is less than about 1 micrometer. The nanoparticles can be coated with an inorganic or organic layer and can be surface-modified. The nanoparticles can be used in many biological assays.

FIELD OF THE INVENTION

This invention relates to hybrid nanoparticles and process of makingthem. More particularly, the present invention relates tonanometer-sized magnetically responsive fluorescent particles andprocess of making them.

BACKGROUND OF THE INVENTION

Bio-magnetic particles are surface-modified magnetically responsivemicrospheres which are widely used in immunoassays, gene engineering,cell separation etc. Roy A. Whitehead and Lee Josephson et al provided aprocess for the preparation of magnetic particles to which a widevariety of molecules might be coupled. The particles comprisedferromagnetic, superparamagnetic or paramagnetic oxides of iron, cobaltor nickel as a metal oxide core, generally surrounded by an adsorptivelyor covalently bound sheath or coat bearing organic functionalities towhich bio-affinity adsorbents might be covalently coupled. Magneticparticles are useful in biological separations in radioimmunoassay, cellisolation, affinity chromatography, immobilized enzyme systems, nucleicacid hybridization, and other biological systems. See U.S. Pat. No.4,554,088, U.S. Pat. No. 4,672,040, U.S. Pat. No. 4,628,037.

Fluorescence immunoassay (FIA) is also widely used in biotechnology.There are many fluorescent materials that can be used as fluorescentlabels, such as organic fluorescent dyes, quantum dots, down-convertingrare-earth phosphor nanoparticles and up-converting phosphornanoparticles etc.

Wang et al provided a process of preparing magnetically responsivefluorescent polymer particles with sizes ranging from 1 to 100 microns.The fluorescent material was used to determine the number of theparticles. These polymer particles comprise polymeric core particlescoated evenly with a layer of polymer containing magnetically responsivemetal oxide as highly sensitive quantitative reagents for biochemicaland immunological studies. The surface of these magnetically responsivepolymer particles can be coated further with another layer offunctionalized polymer. These magnetically responsive fluorescentpolymer particles can be used for passive or covalent coupling ofbiological material such as antigens, antibodies, enzymes or DNA/RNAhybridization and used as solid phase for various types of immunoassay,DNA/RNA hybridization probes assay, affinity purification, cellseparation and other medical, diagnostic, and industrial applications.See U.S. Pat. No. 6,013,531.

Chandler et al provided a process of preparing magnetically-responsivefluorescently-tagged particles. These hybrid microspheres areconstructed using fluorescent or luminescent microspheres and magneticnanoparticles. Reactive moieties on the surface of the resultantparticles can be used for attachment of biologically active molecules,thus allowing selective separations and analytical assays to beperformed. Distinguishable subsets of microspheres can be constructedbased on fluorescent intensities, and separations can be affected basedon variable degree of magnetic content. Multiple particles populationsthus constructed will find utility in a number of fields, includingclinical biological assays See U.S. patent application Ser. No.09/826,960.

BRIEF SUMMARY OF THE INVENTION

The present invention provides nanometer-sized fluorescent magneticparticles and processes of preparing the nanometer-sized fluorescentmagnetic particles. These nanoparticles can be coated with an inorganicor organic layer and can be surface-modified. These nanoparticles can beconstructed using fluorescent materials such as fluorescent dyes,fluorescent quantum dots, down-converting rare-earth phosphors, orup-converting phosphors. The magnetic materials for these nanoparticlescan be superparamagnetic, paramagnetic, ferromagnetic metal oxidenanoparticles such as Fe₃O₄, γ-Fe₂O₃, or other oxides of cobalt, nickel,or manganese. These nanoparticles can be used as both solid phasecarrier which can be manipulated by a magnet and fluorescent labels forvarious types of immunoassay, DNA/RNA hybridization, affinitypurification, cell separation and other medical, diagnostic, andindustrial applications.

In one aspect, the present invention is directed to a nanoparticlecomprising a core particle, wherein the core particle comprises amagnetic material and a fluorescent material, and wherein thenanoparticle has a particle size less than about 1 micrometer.

In another aspect, the present invention is directed to a process ofpreparing a nanoparticle comprising a magnetic particle coated with aphosphor fluoride, which process comprises: a) dispersing ananometer-sized magnetic particle and an aqueous fluoride-containingcompound in de-ionized water; b) contacting the mixture of step a) withan aqueous solution containing soluble salts of a phosphor host, anabsorber/emitter pair, and a rare-earth metal chelator by stirring for asufficient time to allow formation of a phosphor fluoride precipitatewhich forms a coating around the magnetic particle; and c) heating themagnetic particle with the phosphor fluoride coating of step b) at atemperature ranging from about 300° C. to about 450° C. for a period oftime ranging from about 1 hour to about 10 hours to obtain the phosphorfluoride coated magnetic particle that emits light in the visiblewavelength range when excited by long wavelength light.

In another aspect, the present invention is directed to a process ofpreparing a nanoparticle comprising fluorescent particles and magneticparticles coated with silica, which process comprises: a) dispersingnanometer-sized magnetic particles and nanometer-sized fluorescentparticles in an alcohol; b) adding de-ionized water and ammonia having aconcentration of about 28% (w/w) to the mixture of step a) at atemperature ranging from about 20° C. to about 80° C.; and c) stirringthe mixture of step b) after adding n-ethyl silicate (TEOS) for a periodof time ranging from about 0.5 hour to about 8 hours to obtain thenanoparticle.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 illustrates a nanometer-sized particle having a three-layerstructure. The black area can be a nanometer-sized magnetic particle orfluorescent particle. The grey area can be a fluorescent material or amagnetic material layer. The shaded area is an outer shell.

FIG. 2 illustrates a nanometer-sized particle having a core composed ofmagnetic material and fluorescent material and a shell (shaded). Thecore can be a fluorescent material (black) doped with a magneticmaterial (grey) or a magnetic material (black) doped with a fluorescentmaterial (grey).

FIG. 3 illustrates a nanometer-sized particle comprising a fluorescentnanosphere (black), a magnetic nanosphere (grey), and a material(shaded) to bind them together.

FIG. 4 illustrates a 200,000 times transmission electron micrograph(TEM) of fluorescent magnetic particles prepared by the processdescribed in Example 1.

FIG. 5 illustrates an 80,000 times transmission electron micrograph(TEM) of fluorescent magnetic particles prepared by the processdescribed in Example 1.

FIG. 6 illustrates another 80,000 times transmission electron micrograph(TEM) of fluorescent magnetic particles prepared by the processdescribed in Example 1.

FIG. 7 illustrates magnetic hysteresis loop of fluorescent magneticparticles prepared by the process described in Example 1.

FIG. 8 illustrates emission curve of up-converting fluorescence (excitedat 980 nm) of fluorescent magnetic particles prepared by the processdescribed in Example 1.

FIG. 9 illustrates emission curve of down-converting fluorescence ofCasein⁺ fluorescent magnetic particles prepared by the process describedin Example 2.

FIG. 10 illustrates a 50,000 times transmission electron micrograph(TEM) of three-layer fluorescent magnetic particles prepared by theprocess described in Example 8.

FIG. 11 illustrates a 100,000 times transmission electron micrograph(TEM) of three-layer fluorescent magnetic particles prepared by theprocess described in Example 8.

FIG. 12 illustrates emission curve of up-converting fluorescence(excited at 980 nm) of three-layer fluorescent magnetic particlesprepared by the process described in Example 8.

FIG. 13 illustrates magnetic hysteresis loop of three-layer fluorescentmagnetic particles prepared by the process described in Example 8.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “phosphor” means solid, inorganic, crystalline materialthat shows luminescence upon optical excitation.

As used herein, “phosphor host” means one (and usually the major one) ofthe three components in a phosphor which does not participate in thelight absorption or light emission process, but provides structuralenvironment for the other two components.

As used herein, “absorber/emitter pair” means the two components in aphosphor which respectively absorbs longer-wavelength light and emitsshorter-wavelength light to complete the up-converting process.

B. Fluorescent Magnetic Nanoparticles

The present invention relates to nanometer-sized hybrid fluorescentmagnetic particles and processes of making them. The particles are ofnanometer-scale and are magnetically-responsive hybrid microspheres thatcan emit fluorescence when excited by light. The invention can findtheir utility in a number of fields, including bio-separation anddetection.

In one aspect, the present invention provides a nanoparticle comprisinga core particle, wherein the core particle comprises a magnetic materialand a fluorescent material, and wherein the nanoparticle has a particlesize less than about 1 micrometer.

The nanoparticle of the invention may have a size less than about 750nm, about 500 nm, or about 300 nm. In some embodiments, the particlesize ranges from about 35 nm to about 200 nm. In some embodiments, theparticle size ranges from about 80 nm to about 200 nm.

Exemplary magnetic material includes a superparamagnetic material, aparamagnetic material, and a ferromagnetic material. In someembodiments, the magnetic material is a metal oxide, such as, oxide ofcobalt, nickel, manganese, and iron. In a specific embodiment, the metaloxide is Fe₃O₄. In another embodiment, the metal oxide is γ-Fe₂O₃.

In some embodiments, the saturation magnetization of the nanoparticle isbetween about 5 emu/g to about 60 emu/g.

Exemplary fluorescent material includes a fluorescent dye, a fluorescentorgano-metallic compound, an up-converting fluorescent phosphor, adown-converting fluorescent phosphor, and a fluorescent quantum dot. Theup-converting fluorescent material can be a phosphor fluoride, such as aphosphor fluoride having a formula of YF₃:Yb,Er or NaYF₄:Yb,Er. In someembodiments, the up-converting phosphor contains molybdenum. Thedown-converting phosphor can have a formula of CaS:Eu³⁺ or SiAlO₂:Eu³⁺.The fluorescent quantum dot of the inventions can be CdSe/CdS, ZnS/CdSe,or GaAs.

The nanoparticle of the invention can have different configurations forthe core particle. For example, the core particle of the invention cancomprise a fluorescent nanometer-sized particle covered by a layer of amagnetic material. The fluorescent nanometer-sized particle can be apolymer or silica particle containing a fluorescent material.Alternatively, the core particle can comprise a magnetic particlecovered by a layer of a fluorescent material. In another configuration,the core particle can comprise fluorescent particles doped with amagnetic material or magnetic particles doped with the fluorescentmaterial. In another configuration, core particle comprises a magneticparticle, a fluorescent particle, and a material to bind the magneticparticle and the fluorescent particle together. The binding material canbe SiO₂. In some embodiments, the core particle has a coating layer. Forexample, the core particle can be coated with SiO₂. The thickness of thecoating layer may vary, and the size of the nanoparticle and saturationmagnetization may depend on the thickness of the coating layer.

In some embodiments, the surface of the nanoparticle can be modified tocomprise a functional group. Exemplary functional group includes —COOH,—CHO, —NH₂, —SH, —S—S—, an epoxy group, and a trimethoxysilyl group. Insome embodiments, the functional group is immobilized on the surface thenanoparticle.

In some embodiments, a bio-molecule may be linked or conjugated to thenanoparticle, covalently or non-covalently. Exemplary bio-moleculeincludes an amino acid, a peptide, a protein, a nucleoside, anucleotide, an oligonucleotide, a nucleic acid, a vitamin, amonosaccharide, an oligosaccharide, a carbohydrate, a lipid and acomplex thereof. The bio-molecule may be conjugated to the nanoparticlevia a chemical functional group or via binding to another biomolecule(such as biotin, streptavidin, and protein A) contained on the surfaceof the nanoparticle.

The nanoparticle can have any suitable shape, e.g., a rectangle, acircle, an ellipse, or other regular or irregular shapes. Preferably,the nanoparticle has a spherical shape.

C. Processes of Preparing Fluorescent Magnetic Nanoparticles

In another aspect, the present invention is directed to a process ofpreparing a nanoparticle comprising a magnetic particle coated with aphosphor fluoride, which process comprises: a) dispersing ananometer-sized magnetic particle and an aqueous fluoride-containingcompound in de-ionized water; b) contacting the mixture of step a) withan aqueous solution containing soluble salts of a phosphor host, anabsorber/emitter pair, and a rare-earth metal chelator by stirring for asufficient time to allow formation of a phosphor fluoride precipitatewhich forms a coating around the magnetic particle; and c) heating themagnetic particle with the phosphor fluoride coating of step b) at atemperature ranging from about 300° C. to about 450° C. for a period oftime ranging from about 1 hour to about 10 hours to obtain the phosphorfluoride coated magnetic particle that emits light in the visiblewavelength range when excited by long wavelength light.

The nanometer-sized magnetic particle and the aqueousfluoride-containing compound can be dispersed in the de-ionized water byany suitable methods, e.g., sonication.

The present process can further comprise coating the phosphor fluoridecoated magnetic particle of step c) with a coating layer, e.g., a SiO₂layer. The surface of the nanoparticle can be further modified tocomprise a functional group. Exemplary functional group includes —COOH,—CHO, —NH2, —SH, —S—S—, an epoxy group, and a trimethoxysilyl group.

The present process can further comprise immobilizing any desirablemoieties, e.g., biological molecules, to the phosphor fluoride particle.For example, the present process can further comprise immobilizing abiological molecule to the SiO₂-protected particle. Any suitablebiological molecule can be used. Exemplary biological molecules includean amino acid, a peptide, a protein, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a vitamin, a monosaccharide, anoligosaccharide, a carbohydrate, a lipid and a complex thereof.

Any suitable phosphor host can be used in the present processes. Forexample, yttrium, lanthanum or gadolinium can be used as the phosphorhost in the present processes. Any suitable absorber can be used in thepresent processes. For example, ytterbium can be used as the absorber inthe present processes. Any suitable emitter can be used in the presentprocesses. For example, erbium, holmium, terbium or thulium can be usedas the emitter in the present processes. In a specific embodiment, theabsorber is ytterbium and the emitter is erbium, holmium, terbium orthulium.

Any suitable rare-earth metal chelator can be used in the presentprocesses. For example, ethylenediamineteraacetic acid,triethylenetetraaminhexaacetic acid, diethylenetriaminepentaacetic acid,hydroxyethylethylenediaminetriacetic acid,1,2-diaminocyclohexanetetraacetic acid, ethylene glycol bis(b-aminoethylether) tetraacetic acid and a salt thereof can be used asthe rare-earth metal chelator in the present processes.

Any suitable aqueous fluoride-containing compound can be used in thepresent processes. For example, NaF, KF, NH₄F and HF can be used as theaqueous fluoride-containing compound in the present processes. Theaqueous fluoride-containing compound can be contained in an aqueoussolution prior to or concurrently with contacting with the preparedaqueous solution of soluble salts of the phosphor host, theabsorber/emitter pair and the rare-earth metal chelator. The amount ofthe rare-earth metal chelator and the amount of total rare-earth ions inthe aqueous solution can have any suitable ratios. For example, theamount of the rare-earth metal chelator can be about 0-1 times theamount of total rare-earth ions in the aqueous solution.

The soluble salts of the phosphor host and the absorber/emitter pair canbe prepared by any suitable methods. For example, the soluble salts ofthe phosphor host and the absorber/emitter pair can be obtained bydissolving the corresponding metal oxide in hydrochloric acid or nitricacid and subsequently removing the residual acid.

In another aspect, the present invention is directed to a process ofpreparing a nanoparticle comprising fluorescent particles and magneticparticles coated with silica, which process comprises: a) dispersingnanometer-sized magnetic particles and nanometer-sized fluorescentparticles in an alcohol; b) adding de-ionized water and ammonia having aconcentration of about 28% (w/w) to the mixture of step a) at atemperature ranging from about 20° C. to about 80° C.; and c) stirringthe mixture of step b) after adding n-ethyl silicate (TEOS) for a periodof time ranging from about 0.5 hour to about 8 hours to obtain thenanoparticle.

Any nanometer-sized magnetic particles can be used for the presentprocess. Exemplary magnetic particles includes superparamagnetic,paramagnetic, and ferromagnetic nanometer-sized particles, andnanometer-sized magnetic oxide of cobalt, nickel, and manganese.

The fluorescent particles of the invention can have a formula ofYF₃:Yb,Er. The fluorescent particles can also have a formula ofNaYF4:Yb,Er. The fluorescent particle can be a fluorescein-doped silicaparticle.

The surface of the nanoparticle can be modified to contain a functionalgroup. Exemplary functional group includes —COOH, —CHO, —NH₂, —SH,—S—S—, an epoxy group, and a trimethoxysilyl group.

The present process can further comprise immobilizing any desirablemoieties, e.g., biological molecules, to the phosphor fluoride particle.For example, the present process can further comprise immobilizing abiological molecule to the SiO₂-protected particle. Any suitablebiological molecule can be used. Exemplary biological molecules includean amino acid, a peptide, a protein, a nucleoside, a nucleotide, anoligonucleotide, a nucleic acid, a vitamin; a monosaccharide, anoligosaccharide, a carbohydrate, a lipid and a complex thereof. In someembodiments, the alcohol used in the process is 3-propanol.

In other embodiments, the nanometer-sized magnetic particles and thenanometer-sized fluorescent particles are dispersed in the alcohol bysonication for a period of time ranging from about 0.5 hour to about 1hour.

D. Exemplary Embodiments

The present invention relates to nanometer-sized hybrid fluorescentmagnetic particles and processes of making them. Some embodiments of thenanoparticles of the invention and the processes of making them aredescribed in more detail below and in the Examples.

In some embodiments, the nanoparticles of the invention have athree-layer structure shown in FIG. 1. The black area in the figure is ananometer-sized magnetic particle. The grey area in the figure is afluorescent material layer with nanometer-thickness, such as inorganic,organic, or polymer fluorescent materials. The outer shell can beinorganic materials such as silica, or polymeric organic compounds.

Alternatively, the black area is a nanometer-sized fluorescent particle,including inorganic, organic or polymer fluorescent particles. The greyarea is a magnetic material layer with nanometer-thickness. The magneticmaterial can be a hybrid material made of magnetic materials and polymeror some other materials. The outer shell can be inorganic materials suchas silica, or polymeric organic compounds.

The magnetic materials can be superparamagnetic, paramagnetic orferromagnetic nanometer-sized particles or other nanometer-sizedmagnetic oxide of iron, cobalt, nickel or manganese etc.

The fluorescent materials can be up-converting fluorescent materials forexample, phosphor fluoride nanoparticles (Yi Guangshun et al, CN:02116679.X), molybdenum up-converting phosphor particles (Yi Guangshunet al, CN 01134861.5), etc.; nanometer-sized down-converting rare-earthmaterials, for example, CaS:Eu3+, SiAlO2:Eu3+, etc; fluorescent quantumdots, for example, CdSe/CdS, ZnS/CdSe, GaAs, etc.; and fluorescentnanometer-sized particles, for example, polymer nanometer-sizedparticles containing fluorescent materials (Taylor J R et al Anal.Chem., 2000, 72: 1979-1986) and luminophore-doped silica nanoparticles(Santra S et al, Anal. Chem., 2001, 73: 4988˜4993), etc.

Referring to Yi's patent (CN: 02116679X), a type of three-layerup-converting fluorescent magnetic nanoparticles can be prepared in thefollowing way. Nanometer-sized magnetic particles and an aqueousfluoride-containing compound is dispersed in de-ionized water bysonication. An aqueous solution of soluble salts of a phosphor host, anabsorber/emitter pair and a rare-earth metal chelator are added into thesolution. After vigorous stirring for sufficient time the precipitate ofphosphor fluoride is formed as a coating around the magnetic particles.The two-layer structure is formed. Then the particles with a precipitateof phosphor fluoride layer are heated at a temperature ranging fromabout 300° C. to 450° C. for a period of time ranging from about 1 hourto about 10 hours to obtain two-layer magnetic particles with shells ofphosphor fluoride materials that emit light in the visible wavelengthrange when excited by infrared ray. These uniform particles have a sizeof less than 150 nm. A three-layer structure might be formed bicoating asilica layer or polymer layer around the two-layer fluorescent magneticparticle. Furthermore these three-layer particles might besurface-modified with different groups. The particles have a size lessthan 300 nm. Process is further described in detail in Example 8.

These fluorescent magnetic particles having surface modification withamino group, epoxy group, trimethoxysilyl group, sulfhydryl group andother functional groups can be obtained by reacting with3-aminopropy-trimethoxyklne (APS), 3-glycidoxypropyl-trimethoxysilane(GPMS), g-methacryloxypropyltrimethoxy-silane (MPS),3-mercaptopropyltrimethoxysilane (MPTS) and other functional silanereagents respectively. The epoxy-modified particles can be then treatedsequentially with 0.01 mol/L hydrochloric acid and 0.2 mol/L sodiumperiodate solution to obtain aldehyde modified particles.

In some embodiments, the nanoparticle of the invention is ananometer-scaled sphere with a core-shell structure (FIG. 2), in whichthe core has uniform composition composed of magnetic and fluorescentmaterials. The shell is a protective coating of inorganic or polymericmaterial with surface active functionalities.

The core can be a fluorescent material doped with a magnetic material ora magnetic material doped with a fluorescent material.

The magnetic materials can be superparamagnetic, paramagnetic,ferromagnetic nanometer-sized materials, or other nanometer-sizedmagnetic oxide of iron, cobalt, nickel or manganese.

The fluorescent materials can be up-converting fluorescent materials forexample, phosphor fluoride nanoparticles (Yi Guangshun et al, CN:02116679.X), molybdenum up-converting phosphor particles (Yi Guangshunet al, CN 01134861.5); nanometer-sized down-converting rare-earthmaterials, for example, CaS:Eu3+, SiAlO2:Eu3+; fluorescent quantum dots,for example, CdSe/Cd, ZnS/CdSe, GaAs; and fluorescent nanometer-sizedparticles, for example, polymer nanometer-sized particles containingfluorescent materials (Taylor J R et al Anal. Chem., 2000, 72:1979-1986) and luminophore-doped silica nanoparticles (Santra S et al,Anal. Chem., 2001, 73: 4988˜4993).

These fluorescent magnetic particles having surface modification withamino group, epoxy group, trimethoxysilyl group, thiohydroxy group andother functional groups can be obtained by reacting with3-aminopropy-trimethoxysilne (APS), 3-glycidoxypropyl-trimethoxysilane(GPMS), g-methacryloxypropyltrimethoxy-silane (MPS),3-mercaptopropyltrimethoxysilane (MPTS) and other functional silanereagents respectively. The epoxy-modified particles can be then treatedsequentially with 0.01 mol/L hydrochloric acid and with 0.2 mol/L sodiumperiodate solution to obtain aldehyde modified particles.

In other embodiments, the nanoparticle of the invention comprises afluorescent nanosphere, a magnetic nanosphere and a material to bindthem together (FIG. 3).

Nanometer-sized magnetic particles and fluorescent nanoparticles can bedispersed in 3-propanol by sonication for a period of time ranging fromabout 0.5 hour to about 1 hour. De-ionized water and 28% ammonia(NH₃.H₂O) are added at a temperature ranging from about 20° C. to 80° C.Then, n-Ethyl silicate (TEOS) is added and the mixture is stirred for aperiod of time ranging from about 0.5 hour to 8 hours. The fluorescentmagnetic particles coated with silica are obtained

To obtain surface-modified nanoparticles, various silanes can be addedinto the reaction mixture described above and allowed to react for aperiod of time. The nanoparticles are separated with a magneticconcentrator (commercially acquired from ProMega Co.) and washed with3-propanol, distilled water and ethanol. The nanoparticles are thendried at temperature from 40° C. to 110° C. Nanoparticles having surfacemodification with various of groups are obtained.

The magnetic materials can be superparamagnetic, paramagnetic orferromagnetic nanometer-sized particles or other nanometer-sizedmagnetic oxide of iron, cobalt, nickel or manganese etc.

The fluorescent materials include up-converting fluorescent materialsfor example, phosphor fluoride nanoparticles (Yi Guangshun et al, CN:02116679.X), molybdenum up-converting phosphor particles (Yi Guangshunet al, CN 01134861.5); nanometer-sized down-converting rare-earthmaterials, for example, CaS:Eu³⁺, SiAlO2:Eu³⁺; fluorescent quantum dots,for example, CdSe/CdS, ZnS/CdSe, GaAs; and fluorescent nanometer-sizedparticles, for example, polymer nanometer-sized particles containingfluorescent materials (Taylor J R et al Anal. Chem., 2000, 72:1979-1986) and luminophore-doped silica nanoparticles (Santra Set al,Anal. Chem., 2001, 73: 4988˜4993).

The material which binds fluorescent materials and magnetic materialstogether can also be polymeric organic compounds besides silicadescribed above.

Fluorescent magnetic nanoparticles having surface-modification withamino group, epoxy group, trimethoxysilyl group, sulfhydryl group andother functional groups can be obtained by reacting with3-aminopropy-trimethoxysilne (APS), 3-glycidoxypropyl-trimethoxysilane(GPMS), g-methacryloxypropyltrimethoxy-silane (MPS),3-mercaptopropyltrimethoxysilane (MPTS) and other functional silanereagents respectively. The epoxy-modified particles can be then treatedsequentially with 0.01 mol/L hydrochloric acid and with 0.2 mol/L sodiumperiodate solution to obtain aldehyde modified particles.

The nanometer-sized fluorescent magnetic particles prepared in theprocess described above may have the following characteristics:

1) The particle size is on the nanometer scale and can range from about50 nm to about 200 nm. The particle size depends on the size of coresand the thickness of the coating layer.

2) The particles can have saturation magnetization ranging from about 5emu/g to about 60 emu/g. The saturation magnetization can vary with thethickness of the coating layer. The particles can have a very lowcoercivity (for example, less then 20G). All these features meet therequirement for bio-separation.

3) The particles which embed inorganic nanometer-sized fluorescentmaterial such as quantum dots or up-converting fluorescent materialshave better fluorescent characteristic, larger Stokes shift, lowerbackground and greater signal-noise ratio than using fluorescent dyes.

4) The particles can be coated with amino group, epoxy group, aldehydegroup, or other function groups which can be used to link proteins,nucleic acid, and other biomolecules to the surface of the particles.

5) These nanoparticles can be used both as solid phase carrier inseparation and fluorescent labels for various types of immunoassay,DNA/RNA hybridization and detection, affinity purification, cellseparation and other medical, diagnostic, and industrial applications.

E. Examples Example 1 Preparation of Fluorescent Magnetic Nanoparticles

1) To prepare nanometer-sized magnetic particles, 1.622 g of ferricchloride (FeCl₃) and 5.560 g of ferrous sulfate (FeSO₄.7H₂O) weredissolved in 200 ml of de-ionized water which had been de-oxygenizedwith nitrogen bubble overnight. The concentration of ferric ion ([Fe³⁺])was 0.05 mol/L and the concentration of ferrous ion ([Fe²⁺]) was 0.10mol/L. 1.0 g of polyglycol-4000 was added in the mixture and dispersedby sonication for 30 minutes. The mixture was stirred rapidly at atemperature of 60° C. Then 10 ml of 28% ammonia was added into themixture rapidly. The mixture was stirred rapidly for 30 minutes withnitrogen atmosphere all the time. The superparamagnetic particles wasisolated with a magnetic concentrator and washed several times withde-ionized water and alcohol. The particles were dried in vacuum at atemperature of 60° C. overnight. Dark brown nanometer-sized magneticparticles were obtained after pestling.

2) 15 mg of magnetic particles prepared in step 1) and 15 mg ofYF3:Yb,Er prepared according to CN:02116679.X were dispersed in 100 mlof 3-propanol by sonication for more than 30 minutes so that two typesof particles were well-distributed in the solution. 8.94 ml of 28%ammonia was then added into the mixture as a catalyzer and 7.5 ml ofde-ionized water was added as a hydrolytic reagent. The mixture washeated at temperature of 40° C. in the oil bath. Then, 0.2 ml of n-ethylsilicate (TEOS) was added into the mixture and after stirred for 3hours. The particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water and alcohol. Thefluorescent magnetic particles prepared were dried in vacuum at atemperature of 110° C. for 6 hours.

Example 2 Preparation of Magnetic Particles with Red Fluorescence

1) To prepare magnetic nanoparticles with red fluorescence, 30 mg ofmagnetic particles prepared in example 1 and 30 mg of nanometer-sizedrare-earth particles CaS:Eu³⁺ were dispersed in 80 ml of 3-propanol bysonication for more than 30 minutes. The mixture turned to bewell-distributed solution after enough sonication. 8.94 ml of 28%ammonia was added into the mixture as a catalyzer and 7.5 ml ofde-ionized water was added as a hydrolytic reagent. The mixture washeated at temperature of 40° C. in the oil bath. Then, 0.15 ml ofn-ethyl silicate (TEOS) was added into the mixture and the reaction wasstopped after stirring for about 3 hours.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water, and alcohol.Then the particles were dried in vacuum at a temperature of 60° C. for 5hours. The magnetic particles with red fluorescence were obtained.

Example 3 Preparation of Fluorescent Magnetic Nanoparticles with AminoGroups on the Surface

1) To obtain fluorescent magnetic nanoparticles with amino groups on thesurface, 30 mg of magnetic particles prepared in Example 1 and 20 mg ofYF3:Yb,Er prepared according to CN:02116679.X were dispersed in 80 ml of3-propanol by sonication for more than 30 minutes so that the particleswere well-distributed in the solution. 8.94 ml of 28% ammonia was addedinto the mixture as a catalyzer and 7.5 ml of de-ionized water was addedas a hydrolytic reagent. The mixture is heated at temperature of 40° C.in the oil bath. Then, 0.1 ml of n-ethyl silicate (TEOS) was added intothe mixture and the mixture was stirred for about 2 hours. Then, 0.1 mlof 3-aminopropy-trimethoxysilne(APS) was added into the mixture. Thereaction continued for one additional hour.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water and alcohol. Thefluorescent magnetic particles were then dried in vacuum at atemperature of 50° C. for 6 hours. Fluorescent magnetic nanoparticleswith amino groups on the surface were obtained.

Example 4 Preparation of Fluorescent Magnetic Nanoparticles withAldehyde Groups on the Surface

1) To obtain fluorescent magnetic nanoparticles with aldehyde groups onthe surface, 30 mg of magnetic particles prepared in Example 1 and 30 mgof YF3:Yb,Er prepared according to CN:02116679.X were dispersed in 160ml of 3-propanol by sonication for 30 to 40 minutes so that theparticles were well-distributed in the solution. 17.8 ml of 28% ammoniaaqueous was added into the mixture as a catalyzer and 15.0 ml ofde-ionized water was added as a hydrolytic reagent. The mixture washeated at temperature of 40° C. in the oil bath. 0.1 ml of n-ethylsilicate (TEOS) was then added into the mixture. After being stirred forabout 2 hours, the reaction was stopped.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water, and alcohol.After being washed with acetone. 3 ml toluene solution with 10%3-glycidoxypropyl-trimethoxysilane(GPMS) was mixed with the particlesand the reaction was carried out on the shaking table overnight. Thenthe particles were isolated with a magnetic concentrator and washedseveral times with toluene and acetone. Then the particles were dried invacuum at a temperature of 110° C. for 2 hours.

3) The dried particles were acidified with 4 ml of 0.01 mol/Lhydrochloric acid and the mixture was stirred on a shaking table for 0.5hour. Then the particles were washed with de-ionized water severaltimes. The particles were oxidized with 4 ml sodium periodate solutionat a concentration of 0.2 mol/L for 1 hour. Then the particles wereisolated with a magnetic concentrator and washed with de-ionized water.The particles were dried in vacuum at a temperature of 60° C. for 6hours. The fluorescent magnetic nanoparticles with aldehyde groups onthe surface were obtained.

Example 5 Preparation of Fluorescent Magnetic Nanoparticles with EpoxyGroups on the Surface

1) To obtain fluorescent magnetic nanoparticles with epoxy groups on thesurface, 30 mg of magnetic particles prepared in Example 1 and 30 mg ofYF3:Yb,Er prepared according to CN:02116679.X were dispersed in 160 mlof 3-propanol by sonication for more than 30 minutes so that theparticles were well-distributed in the solution. 17.8 ml of 28% ammoniawas added into the mixture as a catalyzer and 15.0 ml of de-ionizedwater was added as a hydrolytic reagent The mixture was heated attemperature of 40° C. in the oil bath. 0.1 ml of n-ethyl silicate (TEOS)was added into the mixture. After stirring for about 2 hours, 0.1 ml of3-glycidoxypropyl-trimethoxysilane(GPMS) was added into the mixture. Thereaction continued for one additional hour and then was stopped.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water, and alcohol.Then the fluorescent magnetic particles were dried in a vacuum at atemperature of 60° C. for 6 hours. The fluorescent magnetic particleswith epoxy groups on the surface were obtained.

Example 6 Preparation of Fluorescent Magnetic Nanoparticles with a ThinCoating Layer

1) To obtain fluorescent magnetic nanoparticles with a thin coatinglayer, 30 mg of magnetic particles prepared in Example 1 and 30 mg ofYF3:Yb,Er prepared according to CN:02116679.X were dispersed in 160 mlof 3-propanol by sonication for more than 30 minutes so that theparticles were well-distributed in the solution. 17.8 ml of 28% ammoniaaqueous was added into the mixture as a catalyzer and 15.0 ml ofde-ionized water was added as a hydrolytic reagent. The mixture washeated at temperature of 40° C. in the oil bath. 0.05 ml of n-ethylsilicate (TEOS) was added into the mixture. After stirring for about 2hours, the reaction was stopped.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water, and alcohol. Theparticles were dried in vacuum at a temperature of 60° C. for 6 hours.The fluorescent magnetic nanoparticles with a thin coating layer wereobtained.

Example 7 Preparation of Fluorescent Magnetic Nanoparticles with a ThickCoating Layer

1) To obtain fluorescent magnetic nanoparticles with a thick coatinglayer, 30 mg of magnetic particles prepared in Example 1 and 30 mg ofYF3:Yb,Er prepared according to CN:02116679.X were dispersed in 160 mlof 3-propanol by sonication for more than 30 minutes so that theparticles were well-distributed in the solution. 17.8 ml of 28% ammoniaaqueous was added into the mixture as a catalyzer and 15.0 ml ofde-ionized water was added as a hydrolytic reagent. The mixture washeated at temperature of 40° C. in the oil bath. 0.2 ml of n-ethylsilicate (TEOS) was added in the solution during stirring the mixtureand the reaction continued for about 2 hours.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water, and alcohol. Theparticles were dried in a vacuum at a temperature of 60° C. for 6 hours.The fluorescent magnetic nanoparticles with a relatively thick coatinglayer were obtained.

Example 8 Preparation of Fluorescent Magnetic Nanoparticles HavingThree-Layer Structure

1) 100 mg of magnetic particles prepared in Example 1 and 2.1 g ofsodium fluoride were dissolved in 120 ml of de-ionized water and awell-distributed colloid solution was prepared by sonication for morethan 40 minutes.

2) Stock solutions of YCl₃ (0.2 mol/L), YbCl₃ (0.2 mol/L) and ErCl₃ (0.2mol/L) were mixed at a volume ratio of 16.0 mL:3.40 mL:0.60 mL in a 100mL beaker. The molar ratio of lanthanide ions in the mixture wasY³⁺:Yb³⁺:Er³⁺=80:17:3.

3) 20 mL of 0.2 M EDTA solution was introduced into the solutionprepared in 2). Then the mixture solution was introduced into thesolution prepared in 1) with rapidly stirring for 1 hour.

4) The magnetic particles were isolated with a magnetic concentrator andthe nonmagnetic particles were washed away with de-ionized water untilthe cleaning solution was clear. Then the magnetic particles were heatedin muffle furnace for 5 hr with hydrogen atmosphere all the time. Andmagnetic particles having a two-layer structure were obtained.

5) 30 mg of particles prepared in 4) were dispersed in 80 ml of3-propanol by sonication for more than 30 minutes so that the particleswere well-distributed in the solution. 8.94 ml of 28% ammonia aqueouswas added into the mixture as a catalyzer and 7.5 ml of de-ionized waterwas added as a hydrolytic reagent. The mixture was heated at temperatureof 40° C. in the oil bath. 0.1 ml of n-ethyl silicate (TEOS) was addedinto the mixture. After being stirred for about 3 hours, the reactionwas stopped.

6) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de ionized water and alcohol. Theparticles were dried in a vacuum at a temperature of 110° C. for 6hours. The fluorescent magnetic nanoparticles with a three-layerstructure having a size ranging from about 80 to about 200 nm wereobtained.

Example 9 Preparation of FITC Fluorescent Magnetic Particles withAldehyde Groups on the Surface

1) 30 mg of magnetic particles prepared in Example 1 and 20 mg ofFITC-doped silica nanoparticles were dispersed in 160 ml of 3-propanolby sonication for more than 30 minutes so that the particles werewell-distributed in the solution. 17.8 ml of 28% ammonia aqueous wasadded into the mixture as a catalyzer and 15.0 ml of de-ionized waterwas added as a hydrolytic reagent. The mixture was heated at temperatureof 40° C. in the oil bath. 0.1 ml of n-ethyl silicate (TEOS) was addedin the solution. After being stirred for about 2 hours, 0.1 ml of3-glycidoxypropyl-trimethoxysilane(GPMS) was added into the mixture. Thereaction was allowed to continue for one additional hour and wasstopped.

2) Then the particles were isolated with a magnetic concentrator andwashed several times with 3-propanol, de-ionized water, and alcohol.Then the particles were dried in a vacuum at a temperature of 60° C. for6 hours. The FITC fluorescent magnetic nanoparticles with epoxy group onthe surface were obtained.

3) The dried particles were acidified with 4 ml hydrochloric acid with aconcentration of 0.01 mol/L and the mixture was stirred on a shakingtable for 0.5 hour. Then the particles were washed with de-ionized waterseveral times. The particles were oxidized with 4 ml sodium periodatesolution at a concentration of 0.2 mol/L for 1 hour. Then the particleswere isolated with a magnetic concentrator and washed with de-ionizedwater. The particles were then dried in a vacuum at a temperature of 60°C. for 6 hours. The FITC fluorescent magnetic nanoparticles withaldehyde groups on the surface were obtained.

The above examples are included for illustrative purposes only and arenot intended to limit the scope of the invention. Many variations tothose described above are possible. Since modifications and variationsto the examples described above will be apparent to those of skill inthis art, it is intended that this invention be limited only by thescope of the appended claims.

1. A nanoparticle comprising a core particle, wherein the core particlecomprises a magnetic material and a fluorescent material, and whereinthe nanoparticle has a particle size less than about 1 micrometer. 2-3.(canceled)
 4. The nanoparticle of claim 1, wherein the particle size isless than about 300 nanometers.
 5. (canceled)
 6. The nanoparticle ofclaim 1, wherein the particle size is ranging from about 80 nanometersto about 200 nanometers.
 7. The nanoparticle of claim 1, wherein themagnetic material comprises a superparamagnetic, a paramagnetic or aferromagnetic material.
 8. The nanoparticle of claim 1, wherein themagnetic material comprises a metal oxide.
 9. The nanoparticle of claim8, wherein the metal oxide is selected from the group consisting ofoxide of cobalt, nickel, manganese, and iron. 10-11. (canceled)
 12. Thenanoparticle of claim 1, wherein the saturation magnetization of thenanoparticle is between about 5 emu/g to about 60 emu/g. 13-19.(canceled)
 20. The nanoparticle of claim 1, wherein the fluorescentmaterial is a fluorescent manometer-sized particle.
 21. (canceled) 22.The nanoparticle of claim 1, wherein the core particle comprises amagnetic particle covered by a layer of the fluorescent material. 23.The nanoparticle of claim 1, wherein the core particle comprises afluorescent particle covered by a layer of the magnetic material. 24.The nanoparticle of claim 1, wherein the core particle comprisesfluorescent particles doped with the magnetic material.
 25. Thenanoparticle of claim 1, wherein the core particle comprises magneticparticles doped with the fluorescent material.
 26. The nanoparticle ofclaim 1, wherein the core particle comprises a magnetic particle, afluorescent particle, and a material to bind the magnetic particle andthe fluorescent particle together. 27-31. (canceled)
 32. Thenanoparticle of claim 1, which comprises a bio-molecule.
 33. (canceled)34. The nanoparticle of claim 32, wherein the bio-molecule is selectedfrom the group consisting of an amino acid, a peptide, a protein, anucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a vitamin,a monosaccharide, an oligosaccharide, a carbohydrate, a lipid and acomplex thereof. 35-47. (canceled)
 48. A process of preparing ananoparticle comprising fluorescent magnetic particles coated withsilica, which process comprises: a) dispersing nanometer-sized magneticparticles and nanometer-sized fluorescent particles in an alcohol; b)adding de-ionized water and ammonia having a concentration of 28% to themixture of step a) at a temperature ranging from about 20° C. to about80° C.; and c) stirring the mixture of step b) after adding n-ethylsilicate (TEOS) for a period of time ranging from about 0.5 hour toabout 8 hours to obtain the nanoparticle.
 49. The process of claim 48,wherein the magnetic particles are selected from the group consisting ofsuperparamagnetic, paramagnetic, and ferromagnetic nanometer-sizedparticles, and nanometer-sized magnetic oxide of cobalt, nickel, andmanganese. 50-51. (canceled)
 52. The process of claim 48, wherein thefluorescent particle is a fluorescein-doped silica particle.
 53. Theprocess of claim 48, wherein the surface of the nanoparticle is modifiedto contain a functional group. 54-55. (canceled)
 56. The process ofclaim 48, wherein the nanometer-sized magnetic particles and thenanometer-sized fluorescent particles are dispersed in the alcohol bysonication for a period of time ranging from about 0.5 hour to about 1hour.